Mitochondrial and metabolic dysfunction of peripheral immune cells in multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by the infiltration of inflammatory cells and demyelination of nerves. Mitochondrial dysfunction has been implicated in the pathogenesis of MS, as studies have shown abnormalities in mitochondrial activities, metabolism, mitochondrial DNA (mtDNA) levels, and mitochondrial morphology in immune cells of individuals with MS. The presence of mitochondrial dysfunctions in immune cells contributes to immunological dysregulation and neurodegeneration in MS. This review provided a comprehensive overview of mitochondrial dysfunction in immune cells associated with MS, focusing on the potential consequences of mitochondrial metabolic reprogramming on immune function. Current challenges and future directions in the field of immune-metabolic MS and its potential as a therapeutic target were also discussed.


Introduction
Multiple sclerosis (MS) is a chronic multifocal demyelinating neuroinflammatory disease characterized by progressive neurodegeneration and an autoimmune response [1,2].According to the latest Multiple Sclerosis Atlas, 2.8 million people have MS worldwide [3].MS occurs at a younger age compared to other neurological illnesses [4], with a mean age of diagnosis at 32 years.The prevalence of MS is higher in females, with a female-tomale ratio of 3:1 [5].MS can be divided into three clinical manifestations: the relapsing-remitting (RRMS), the secondary progressive (SPMS) and the primary progressive (PPMS) [3].RRMS is the most common form, and it would experience unpredictable onset of neurological symptoms (relapses) and then complete or partially recovery (remissions).Over time, relapsing-remitting attacks become less frequent and neurological function may gradually deteriorate to SPMS.On the other hand, PPMS patients do not experience attacks or remissions, and symptoms continually worsen [3].
Currently, the diagnosis of MS primarily relies on magnetic resonance imaging (MRI), which detects lesions in two or more areas of the central nervous system (CNS) along with corresponding clinical symptoms [6].Cerebrospinal fluid (CSF) analysis and intrathecal immunoglobulin G production can aid in the diagnosis [7,8].However, there has been currently no reliable or sensitive biomarker in peripheral blood to identify the condition.In addition, the majority of existing medications utilized for the treatment of MS primarily improve the patient's condition and slow the advancement of the disease.Hence, it is imperative to identify early diagnostic markers for diagnosis and efficacious treatments.
Genetic, environmental, and immune factors play significant roles in the development of MS.More than 230 genetic variations associated with a higher risk of developing MS have been identified [9,10].Genome-wide association studies (GWAS) provide biological insights into the genetic susceptibility of MS, suggesting that peripheral immune cells are essential mediators of disease risk variants [11].Some of these variants are related to the aspect of myelin structure or mitochondrial function [12].
A specific genetic factor strongly linked to MS development is a variation in the human leukocyte antigen complex known as HLA-DRB1*15:01.This variation plays a significant role in antigen presentation by antigen-presenting cells (APCs) [10,13].Additionally, environmental influences such as obesity, gut microbiota imbalance, virus infection, and smoking contribute to increased risks of developing MS, with Epstein-Barr virus (EBV) infection increasing the risk by 32-fold [14].These factors may affect mitochondrial biogenesis and functions [15].Moreover, immune dysregulation in MS leads to the infiltration of immune cells into the CNS, triggering demyelination, axonal damage, and neurodegeneration [16].The primary target of immune cell attack is the myelin sheath in the white matter of the CNS [17,18].Local inflammation and demyelination lead to the diffusion of self-antigens, sequestration of myelin, and activation of autoreactive T lymphocytes [19,20].B cells primarily present self-antigens to T lymphocytes through the secretion of self-reactive antibodies [21].Each immune cell type has a unique metabolic profile crucial for its function and maintenance [22].Understanding these metabolic profiles may provide new insights into controlling the immune response in MS.
Mitochondria are double-membrane organelle in eukaryotic cells and they are the main site of cellular aerobic respiration.Mitochondria produce adenosine triphosphate (ATP) through oxidative phosphorylation, which occurs in the inner mitochondrial membrane.The electron transport chain (ETC) complex in this process transfers electrons to O 2 to form reactive oxygen species (ROS).Additionally, other mitochondrial processes such as fatty acid metabolism and tricarboxylic acid cycle (TCA) can also promote ROS production.But if the production of ROS exceeds the physiological limit, it can potentially damage cellular components [23].Mitochondria also play a role in apoptosis, regulation of calcium balance, production of mitochondrial DNA (mtDNA), oxidative phosphorylation (OXPHOS), and mitochondrial metabolism [24,25].Mitochondria significantly contribute to the pathogenesis of MS [26][27][28][29] by: (1) accumulating mutations and repairing mtDNA damage; (2) exhibiting abnormal mitochondrial protein or gene expression; (3) displaying defects in mitochondrial enzyme activity and aging; (4) experiencing disturbances in mitochondrial dynamics and changes in the TCA cycle; (5) initiating mitochondria-mediated apoptosis.Elevated glucose and lactate metabolism in active MS lesions have been revealed through investigations using magnetic resonance spectroscopy and positron emission tomography imaging and immunohistochemistry staining [30,31].Further evidence of increased metabolic activity in active MS lesions is derived from the higher quantity and functioning of mitochondria in responsive axons [32].In chronic inactive areas of MS lesions, both the activity of mitochondrial respiratory chain complex IV and mitochondrial mass increase in demyelinated axons [33].
This review summarized the immunological-metabolic mechanisms involved in mitochondrial dysfunction in peripheral immune cells associated with MS.The relationship between MS and mitochondrial function is discussed, with a focus on disease-modifying therapies (DMTs).The aim is to provide insightful hints for investigating the mitochondrial immune function related to MS.

Autoimmune response
The autoimmune response is considered a key pathogenic mechanisms underlying MS, wherein an abnormal immune response targets self-antigens within the CNS [34].This response is triggered by the activation of autoreactive T cells and B cells, which recognize and attack myelin and other components of the CNS [2,35].Activated myelin-specific CD4 + T cells that react to myelin antigens are primarily responsible for this reaction [36].Myelin antigens, together with HLA class II molecules and accessory molecules on the surface of APCs, reactivate CD4 + T cells in their native environment.The presence of autoantibodies against myelin proteins in the serum and CSF of MS patients indicates that the immune system perceives these proteins as foreign entities and initiates an immune response against them.[37,38].Additionally, the presence of inflammatory cells, such as T cells and B cells within MS lesions demonstrates an ongoing immune response within the CNS [39].Reactivation of MS triggers the release of inflammatory cytokines and soluble mediators, which disrupts the blood-brain barrier (BBB), stimulates chemotaxis, and leads to a larger-scale influx of inflammatory cells into the CNS [40] (Fig. 1A).The experimental autoimmune encephalomyelitis (EAE) model, induced by immunizing animals with myelin-derived proteins (or polypeptides), such as myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and myelin basic protein (MBP), serves as a classic animal model of MS.EAE mice Fig. 1 Pathological manifestations of peripheral immune cells in MS.The pathogenesis of MS goes through three main phases: the autoimmune response (A), the chronic inflammatory response (B), and the demyelinating reaction (C).During the autoimmune reaction stage (A), various immune cells, such as T cells, B cells, and myelin-specific CD4 + T cells, penetrate the brain tissue through the blood-brain barrier (BBB).In the chronic inflammatory response (B), adaptive Th cells, Treg, and B cells release cytokines or interferon-γ and antibodies to contribute to the inflammatory response.Additionally, innate immune macrophages (Mφ), and natural killer (NK) cells secrete substances like histamine, trypsin, ROS, NO, inflammatory cytokines, and Granzyme B, which participate in the inflammatory response.Peripheral immune cells, particularly T cells, B cells, monocytes and Mφ, contribute to the demyelination process in MS through direct interactions with oligodendrocytes, the release of pro-inflammatory molecules, and the production of antibodies against myelin proteins.MS monocytes inhibit the phagocytic capacity of myeline debris, whereas exosomes derived from DCs promote myelination (C) model is characterized by the myelin-reactive CD4 + T cells and B cells [35,41].

Chronic inflammatory response
The chronic inflammatory response in MS is characterized by the activation and proliferation of T cells, B cells, and other immune cells, resulting in the release of proinflammatory cytokines, chemokines, ROS and nitric oxide (NO) [16] (Fig. 1B).This chronic inflammation leads to the destruction of myelin, axons, and oligodendrocytes, ultimately causing neurological dysfunction and disability [42].Recent studies have implicated the innate immune system in the pathogenesis of MS.Activation of toll-like receptors (TLRs) activation leads to the release of pro-inflammatory cytokines and chemokines, contributing to the chronic inflammatory response in MS [43].Pro-inflammatory Th1 and Th17 have been associated with the pathology of MS [44].On the contrary, regulatory T cell (Treg) mediates the dysregulation of T cell response in MS by reducing their number and activity [45,46].T cells and B cells can be detected in damaged white and gray matter [47].In MS, B cells differentiate into memory cells or plasma cells, which subsequently secrete autoantibodies involved in antibody-dependent cellular cytotoxicity and complement damage.B cells also have the ability to secrete pro-inflammatory cytokines, including lymphotoxin (LT) and tumor necrosis factor (TNF)-α, significantly contributing to T cell activation [48].Mucosal-associated invariant T (MAIT) cells, a subset of innate-like CD8 + T cells, express semi-invariant T cell receptors as well as accumulate in the MS brain and produce IL-17 [49].Innate immunity involves mast cells, macrophages (Mφ) and natural killer (NK) cells.Mast cells in MS plaques release histamine and trypsin simultaneously to facilitate BBB opening [16].Mφ produce cytokines such as interleukin (IL)-12, IL-23, and are also involved in the clearance of demyelinating debris [16].Some subgroups of microglia and Mφ produce TNF-α, ROS and NO in the activated state, exerting direct neurotoxic effects.CD56 bright NK cells promote the production of granzyme B, which is involved in cytotoxicity and autologous CD4 + T cells proliferation [50].

Demyelination reaction
Peripheral immune cells are activated and enter the CNS where they contribute to the destruction of the myelin sheath [51] (Fig. 1C).Myelin-specific T cells exacerbate demyelinating lesions in CNS autoimmunity by releasing cytokines such as IL-17, interferon (IFN)-γ, and granulocyte macrophage-colony stimulating factor (GM-CSF) [52,53].Besides, autoreactive T cells mistakenly target and attack myelin, which is the protective covering of nerve fibers in the CNS.These activated T cells release inflammatory cytokines and chemokines that attract other immune cells to the site of inflammation, leading to the further damage to the myelin sheath [54,55].In progressive MS, cortical demyelination is associated both with B cell-rich structures in the meninges and plasma cell accumulation in experimental CNS inflammation [56][57][58], which may be involved in secretory products independent of antibodies, and multiple cytokines produced by B cells in progressive MS patients are cytotoxic to oligodendrocytes and neurons [59,60].On the other hand, autoreactive B cells can differentiate into plasma cells, producing antibodies that bind to the myelin sheath and oligodendrocyte proteins.These bound antibodies result in the induction and activation of the complement proteins on tissue surfaces, directly damaging the myelin sheath and exacerbating demyelination [61,62].Inflamed monocytes derived from individuals with MS exhibit a diminished ability to engulf and remove myelin debris, a process crucial for prompt and effective remyelination [63].Mφ release a substantial quantity of cytokines and NO, penetrating the CNS, which is a vital element in initiating the demyelination response in MS [64].Distinct subgroups of Mφ exert contrasting influences on myelination, with inflammatory Mφ facilitating demyelination [65] and immune-modulatory Mφ facilitating remyelination [66].Dendritic cells (DCs) culture stimulated with low-level IFN-γ-released exosomes can increase myelination and reduce oxidative stress both in vitro and in vivo in EAE mice [67].

Mitochondria in MS T cells
The modulation of T lymphocyte homeostasis in MS is influenced by mitochondrial involvement.Several mitochondrial proteins, including B cell lymphoma 2 (Bcl2), ocular atrophy 1 (OPA1), prohibitin 2 (PHB2), Sirtuin-3 (SIRT3), mitochondrial metalloendopeptidase OMA1, and autophagy related 5 (ATG5), are closely associated with mitochondria-mediated death in MS T cells, probably caused by oxidative stress [29,[68][69][70][71][72].Some studies have shown that T cells from MS patients exhibit altered mitochondrial structure, reduced glycolysis, downregulated expression and activity of OXPHOS subunits, and decreased mitochondrial membrane potential (MMP) [73][74][75][76][77]. Treatment with IFN beta-1α has the potential to restore the diminished glycolysis and mitochondrial respiration activity observed in T cells derived from patients with RRMS.This restorative effect is linked to elevated levels of aldolase, hexokinase 1 (HK-1), enolase 1 (ENO1), glucose transporter 1 (GLUT1), dihydrolipoamide-S-acetyl transferase (DLAT), and dihydrolipoamide-S-succinyl transferase (DLST) production [77].Another study found that during relapse, RRMS patients exhibited increased extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which were not observed during the remission when compared to the healthy controls.Teriflunomide treatment could prevent the proliferation of metabolically active high-affinity T cells involved in the progression of RRMS patients during relapse.This effect is associated with a functional downregulation of OCR and ECAR, along with complex III activity of the ETC in activated T cells.However, it has no impact on mitochondrial content or structure [78].

Mitochondria in MS CD4 + T cells
A study revealed decreased Fas-mediated apoptosis of CD4 + CCR5 + T cells in PPMS patients [79].Moreover, activated CD4 + T cells in patients with RRMS exhibited increased expression of the GLUT1 protein at the plasma membrane.This upregulation facilitated the uptake of glucose, leading to significant glucose metabolism and subsequent lactate production.Oxidative stress may be associated with this process, as it lowers the protective enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX), while raising the expression of the protein Hsp70 [75].Consistently, high glucose intake exacerbates autoimmunity in EAE and promotes the differentiation of Th17 cells through ROS-driven activation of transforming growth factor (TGF)-β signaling in CD4 + T cells.However, there were no changes observed in mitochondrial OCR and ECAR in CD4 + T cell metabolism [80].Tregs with a prominent ROS signature in EAE mice were found.Inhibiting ROS with MitoTEMPO in Tregs of EAE mice attenuated the DNA damage response, prevented Treg cell death, and reduced the differentiation of Th1 and Th17 cells [81].Other findings also indicate that increased ROS during pathogenic Th1 and Th17 cell development could be a potential metabolic target for MS [82].
There were significant differences in lipid species specific to CD4 + T cells between MS patients and control subjects [83].Short-chain fatty acids (SCFAs) and long-chain fatty acids (LCFAs) were shown to be crucial for the differentiation of CD4 + T cells during the progression of EAE mice [84,85].Oleic acid, a type of LCFA, restores suppressive deficiencies in tissueresident Tregs from MS patients by enhancing fatty acid β-oxidation [86].SCFAs, particularly pentanoate, induce metabolic rewiring by increasing mammalian target of rapamycin (mTOR) activity in CD4 + effector T cells.This increase leads to higher acetyl-CoA levels, glucose oxidation, IL-10 secretion, OCR, and suppression of IL-17A production.Ultimately SCFAs affect the balance of Th17/Treg and ameliorate the progress of MS/EAE [87][88][89].Saturated fatty acid side chain phospholipids (PLs) possess natural anti-inflammatory properties, reducing EAE symptoms by inhibiting the activation of autoreactive CD4 + T cells and promoting apoptosis.This effect is achieved through the suppression of Bcl-2-interacting molecule Bim and Bad phosphorylation [90].Deletion of acetyl-CoA carboxylase 1 (ACC1) in CD4 + T cells prevents de novo fatty acid production, inhibits the glycolytic-lipogenic pathway, and protects mice from EAE by reducing the number of IFN-γ + Th17 cells and increasing the number of FOXP3 + Tregs in the spinal cord [91].Atorvastatin, an inhibitor of HMG CoA, inhibits lipid metabolism and suppresses the T cell activation and differentiation of Th1 and Th2 cells.This immune regulation can be reversed by using HMG CoA reductase L-mevalonate [92].These findings offer valuable insights into how lipid metabolism regulates the delicate balance between tolerance and adverse immune responses [93].
Inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which indirectly reduce hypoxia-inducible factor 1α (HIF-1α) activation, have been shown to inhibit Th17 differentiation and exert beneficial effects on MS patients by regulating glycolysis as the main regulator [94].Upregulated ATP-linked OXPHOS in naïve CD4 + T cells can promote Th17 cell differentiation in EAE/MS through decreased expression of aids basic leucine zipper transcription factor TF-like (BATF).Otherwise, this state will switch to Treg differentiation [95].Consistently, mice lacking BATF exhibited resistance to EAE due to increased chromatin accessibility of Th17 transcription factors [96].The upregulation of mitochondrial elongation factor G1 (mEF-G1) is implicated in the mitochondrial translation process, leading to enhanced ETC assembly and an increased intracellular ratio of NAD + /NADH in CD4 + T cells.This upregulation subsequently promotes the development of Th17 and Th1 cells in MS/EAE.The observed phenotypes showed potential for reversal with the administration of the antibiotic linezolid, which perturbing mitochondrial translation in differentiating T cells [97].Defective cellular metabolism, including downregulation of ECAR and OCR, prevents class III phosphatidylinositol 3-kinase (Pik3c3)deficient CD4 + T cells from differentiating into Th1 cells and makes them resistant to EAE induction [98].Deletion of Nur77 enhances OCR and ECAR levels in CD4 + T cells, resulting in Th1 and Th17 differentiation and exacerbating EAE progression [99].Suppression of ATPase activity [95] or the use of PFKFB3 inhibitor [94] or 2-deoxy-glucose (2-DG) [87] to control CD4 + T cell glycolysis can similarly modify Th17/Treg differentiation.Altogether, CD4 + T cells heavily rely on mitochondrial metabolism and dysfunctions for their differentiation in MS disease.The summarized mitochondrial metabolism scheme of MS CD4 + T cells is depicted in Fig. 2.

Mitochondria in MS CD8 + T cells
CD8 + T cells represent another important therapeutic target for MS [100].Cytotoxic CD8 + T cells have been detected in MS plaques, CSF, and demyelinated axons [100].Lactate, a byproduct of glycolysis, accumulates in the CSF of MS patients [101].Several investigations have demonstrated that shifts in the metabolic activities of CD8 + T cells, including glycolysis and OXPHOS, have diverse regulatory effects on the activation, differentiation, and functionality of these immune cells [102].
A higher mitochondrial mass and MMP have been found in the CD8 + T cell subset of RRMS patients, accompanied by increased expression of GLUT1.Treatment with 2-DG, a glucose analogue, leads to reduced activation of CD8 + T cell subsets in RRMS patients, as proved by decreased expression of the early activation marker CD69, reduced levels of the high-affinity IL-2 receptor CD25, and lower production of TNF α [103].
The study demonstrates that dimethyl fumarate (DMF) significantly alters the metabolic profile of human CD4 + and CD8 + T lymphocytes and limits caspase-mediated apoptosis to combat oxidative stress by reducing intracellular glutathione (GSH) levels, which are ROS scavengers.As a result, there is an increase in mitochondrial ROS, MMP levels, and an improvement in Cytc, ultimately resulting in a reduction in mitochondrial OCR [104].Another study has discovered that the inhibition of IL-17-producing CD8 Mφ (Tc17) cells by DMF relies on ROS, which is attributed to heightened PI3K-AKT-T-BET and IL-2-STAT5 signaling pathways.Additionally, DMF-signaling is partially associated with the inhibition of type I or II histone deacetylases (HDACs) and histone acetylation on the IL-17 locus [105].Enhancing our understanding of the role of mitochondria-targeted therapy in MS may offer a potential immunotherapeutic strategy to restrict T cell-mediated autoimmunity in the future.Table 1 summarizes the mitochondrial and metabolic dysfunction mechanisms that influence T cell function.

Mitochondria in MS B cells
B cells can be found in CNS lesions throughout all stages of MS, primarily localized to the perivascular cuffs.
B cells serve as effective APCs expressing costimulatory molecules such as CD40, CD80, and CD86 as well as MHC class II [61,106].Compared to myeloid APCs, they exhibit significantly higher efficiency, approximately 10,000 times, in capturing soluble and membrane-tethered antigens and presenting them to T cells [107].In SPMS patients, mitochondrial damage has been observed around CNS periventricular CD20 + B cells [108].
In CSF memory B cells, single-cell RNA sequencing studies have shown an increase in cholesterol production [109].Obeticholic acid (OCA) is a unique medication that regulates various metabolic processes, including cholesterol production, glucose metabolism, inflammation, and apoptosis [110].It has been proven the effectiveness of OCA in treating EAE, reducing high-density lipoprotein (HDL) levels, cholesterol, and the number of B cells, while OCA alleviates B cell exhaustion by reducing the expression of programmed cell death protein 1
Evidence suggests that increased levels of glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and triosephosphate isomerase (TPI), in the CSF may contribute to B cell clonal growth in the CNS of MS patients [112].
In vitro studies have demonstrated that inhibition of mitochondrial respiration decreases B cell activation (but not glycolysis).On the other hand, both mitochondrial respiration and glycolysis are involved in B cell proliferation, with a notably higher dependence on mitochondrial respiration.Additionally, the expression of costimulatory molecules CD80 and CD86 is reliant on mitochondrial respiration rather than glycolysis.The dysregulated activation and upregulation of CD80 and CD86 on B lymphocytes from untreated individuals with MS can be effectively counteracted by inhibiting Bruton's tyrosine kinase (BTKi) as well as modifying metabolic processes.Furthermore, BTKi therapy reduces OCR in circulating B cells, attenuates their activation and antigen presenting potential in vivo, partially through the PI3K/AKT/ mTOR pathway [113].Taken together, these findings indicate that glycolysis, cholesterol metabolism, and mitochondrial respiration play crucial roles in regulating B cell functions associated with the pathophysiology of MS.Table 2 provides a summary of the effects of mitochondrial and metabolic dysfunction pathways on B cell activities.

Mitochondria in MS monocytes
Monocytes, a type of immune cell, have been shown to influence mitochondrial function in patients with MS.They play a significant role in the demyelination process observed both in MS and EAE and they exhibit high adaptability.Once they infiltrate the CNS during disease progression, monocytes can differentiate into either inflammatory or immune-modulatory macrophages, depending on local conditions.The ratio of these macrophage types ultimately determines the course of MS [114].Functional enrichment analysis using GWAS datasets revealed a strong association among unique risk genes in monocytes and mitochondria and lipid metabolism [11].
The small molecule inhibitor 6877002 targeting the CD40-TRAF6 pathway alters human inflammatory monocytes, resulting in reduced production of ROS, TNF, and IL-6, while increasing the production of IL-10.This treatment also enhances the ability of monocytes to reduce trans-endothelial migration, which is significant when monocytes infiltrate the CNS during neuroinflammation in EAE [115].Lactate dehydrogenase (LDH), consisting of lactate dehydrogenase A (LDHA) and LDHB subunits, is a crucial enzyme in the anaerobic glycolysis metabolic pathway implicated in MS pathology [116].LDHB favors the conversion of lactate to pyruvate, while LDHA, as a rate-limiting enzyme, has a higher affinity for pyruvate, converting it into lactate [117].Remarkably higher levels of LDHA on CD11b + monocytes in blood from EAE mice have been observed compared to the control condition [118].
Monocytes derived from EAE mice treated with 2-DG display decreased glucose uptake and reduced lactate production in the surrounding medium.Additionally, these monocytes exhibited decreased ECAR, which correlated with a decrease in the expression of key glycolytic enzymes including Glut1, HK-2, TPI, pyruvate kinase M (PKM), LDHA, and monocarboxylate transporter (MCT) 1. Monocytes treated with 2-DG adopted an immunemodulatory phenotype, and once them transplanted into EAE mice, resulted in a significant improvement in disease severity [114].These findings indicate that glycolysis is highly upregulated in activated monocytes in MS.
Comparison between the MS group and the control group revealed that monocytes treated with MS-CSF produced more pyruvate and glutamine.Extracellular levels of glutamine, lactate, and pyruvate were significantly increased, while levels of glutamic acid were significantly decreased [119].Quantification of monocyte numbers and assessment of monocytic ROS levels revealed an upregulated trend following DMF administration, allowing for the distinction between responders and non-responders.Mechanistically, a single nucleotide polymorphism (SNP) in NOX3 (rs6919626 A) is associated with ROS production and responsiveness to DMF treatment [120].Mitochondrial dysfunction pathways were upregulated in monocytes of individuals who did Table 2 The impact of mitochondrial and metabolic dysfunction on B cell functioning

Mechanism of mitochondrial and metabolic dysfunction B cell functions References
Downregulated high-density lipoprotein (HDL) production by obeticholic acid (OCA) treatment Reduce the number of B cells and B cell exhaustion [111] Upregulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and trisaccharide phosphoisomerase (TPI) in the CSF Drive B cell clonal expansion [112] BTKi treatment limits OCR in B cells partially through PI3K/AKT/mTOR pathway Attenuate B cell activation and antigen-presenting function [113] not respond to IFN-beta treatment.Monocytic ROS production slightly decreased, and several mitochondrial ETC-related genes showed significant alterations in monocytes that responded to IFN-beta treatment compared to non-responders [121,122].These results suggest that mitochondrial translation, oxidative stress, and intracellular glycolysis are crucial for monocyte function and pathology, particularly in the context of Table 3 Mechanisms of mitochondrial and metabolic dysfunction that affect monocyte functions

Mechanism of mitochondrial and metabolic dysfunction Monocytes functions References
The inhibitor 6877002 decreases ROS production of monocytes through CD40-TRAF6 pathway Reduce pro-inflammatory cytokines (TNF, IL-6) production and trans-endothelial migration ability [115] 2-DG treatment reduces glucose uptake, lactate secretion and ECAR in monocytes, which was correlated with a decrease in the expression of Glut1, HK-2, TPI, PKM, LDHA, and MCT-1 Switch to an anti-inflammatory phenotype and 2-DG treated monocytes attenuate the severity of experimental autoimmune encephalomyelitis (EAE) [114] DMF treatment increases ROS production in monocytes, which was correlated with genetic variation and CpG methylation in monocytic NOX3 Increase the numbers of monocytes [120] IFN-beta treatment alters mitochondrial dysfunction pathway and mitochondrial ETC-related genes Decrease monocytic ROS production [121,122] mitochondrial therapy drugs (Fig. 3).Table 3 provides a summary of the pathways involving mitochondrial and metabolic disorders that impact monocyte activities.

Mitochondria in MS Mφ
Mφ actively participate in the immune response to autoimmune diseases and have been implicated in the destruction of myelin and axons in MS lesions [123].Immunoregulatory Mφ (M2) drive the remyelination of damaged axons, produce neurotrophic factors, and facilitate oligodendrocyte function [64].DMF efficiently inhibits antigen-presenting function in Mφ, suppressing pro-inflammatory mediators such as ROS, inducible NO synthase (iNOS), TNF-α, IL-1β, and IL-6.DMF achieves this by decreasing extracellular signal-regulated kinase (ERK) phosphorylation, promoting M2-like Mφ in an EAE mouse model [124].Furthermore, it has been demonstrated that DMF significantly blocks glycolysis by inhibiting GAPDH in murine Mφ [125].This may help explain how DMF promotes M2-like Mφ, considering that M1 Mφ primarily utilize glycolysis while M2 Mφ exhibit greater OXPHOS activity [125,126].Moreover, FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, inhibits the progression of EAE/MS by upregulating OXPHOS and decreasing glycolysis in Mφ.This leads to the inhibition of Mφ activation and production of pro-inflammatory cytokines, such as TNF and IL-6 [127,128].ROS are rapidly produced in the CNS of MS patients, predominantly by activated Mφ responsible for demyelination and disruption of axons [129,130].These oxidative bursts in Mφ contribute to focal axonal degeneration, which is further exacerbated by mitochondrial damage and iron release from MS lesions [131,132].The p47phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, primarily localized within the zone of initial damage or lesions in patients with acute or early RRMS, accumulates in infiltrating Mφ and mediates ROS generation [133,134].RNA sequencing analysis reveals that fibrin induces the core oxidative stress signature (including neutrophil cytosolic factor (Ncf ) 2, Sod2, and Nox2) and the p47phox subunit of NADPH oxidase expressed by ROS + Mφ [135,136].In an EAE mouse model, the transcriptomic signature of fibrin indicates a decrease in oxidative stress, demyelination, axonal harm, and prevention of paralysis when mice are exposed to the fibrin-targeting antibody 5B8.This evidence suggests that fibrin plays a significant role in triggering gene programs associated with oxidative stress during the activation of peripheral Mφ in EAE [135].Taken together, these findings suggest that the inflammation-associated oxidative burst in Mφ contributes to demyelination and tissue injury mediated by free radicals in the pathogenesis of MS.
MCTs play a crucial role in exporting lactate in glycolytic cells [137].MCT-4 and LDHA are upregulated in inflammatory Mφ of postcapillary venules in MS patients.Knockdown of LDHA or MCT-4 significantly reduces lactate levels in macrophage supernatant after lipopolysaccharide (LPS) treatment.Additionally, the MCT-4 inhibitor α-cyano-4-hydroxy-cinnamic acid (CHCA) effectively decreases lactate levels and prevents inflammatory activity in Mφ.The LDHA inhibitor 3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid (FX11) also exhibits lactatereducing properties in Mφ, inhibiting their inflammatory function [118].Another study shows that enhanced glycolysis in infiltrating MHC-II + Mφ leads to increased expression of glucose transporters (GLUTs) such as GLUT1, GLUT3, GLUT4, and MCT1, actively contributing to demyelination in MS lesion tissue [138].These findings indicate that elevated glycolysis is partially associated with increased expression of glycolytic kinase proteins, which are integral to inflammatory macrophage activation and contribute to demyelination in MS lesions (Fig. 4).The impact of mitochondrial and metabolic dysfunction mechanisms on Mφ functions is summarized in Table 4.

Mitochondria in MS neutrophils
Neutrophils, a crucial population of granulocytes, play a key role in initiating inflammatory responses [139].Neutrophils in MS display an activated phenotype characterized by increased surface expression of TLR-2 and N-formyl-methionyl-leucyl-phenylalanine receptors (fMLPR).Additionally, their adhesion and migration energies are increased [140].Once neutrophils enter the target site and become activated, they exhibit various effector functions aiming at neutralizing the entry of pathogens.These effector activities include phagocytosis, degranulation, and the secretion of ROS.
The abundance and function of mitochondria in neutrophils have been discussed for a long time.It is believed that instead of involved in ATP synthesis primarily, neutrophil mitochondria appear to have diverse functions including mtDNA copy number and mitochondrial potential [141].Moreover, a study found no significant differences in intracellular ROS generation between RRMS patients and healthy controls [142].However, selective deletion of CXCR2 in neutrophils is crucial for downregulating Ncf1.This downregulation leads to the inhibition of ROS and IL-1β production in neutrophils, resulting in attenuated CNS neuronal damage in EAE disease [143].In case of lacking Socs3, neutrophils

Mechanism of mitochondrial and metabolic dysfunction Mφ functions References
DMF blocks the glycolysis process and ROS production by decreasing extracellular signal-regulated kinase (ERK) phosphorylation Inhibit antigen-presenting function in Mφ and switch to M2 phenotype [124,125] FhHDM-1 upregulates oxidative phosphorylation (OXPHOS) and decreases glycolysis Inhibit Mφ activation and pro-inflammatory cytokines expression [127,128] P47phox mediated ROS generation Accumulate in infiltrating Mφ and involved in demyelination [133,134] Fibrin upregulates ROS production through enhancing oxidative stress genes (Ncf2, Sod2, Nox2) and p47phox Promote activation of Mφ and demyelination and axonal damage [135,136] Deleting LDHA or MCT4 reduces lactate production Inhibit pro-inflammatory Mφ activity [118] Glycolysis upregulated in MHC-II + Mφ was associated with glucose transporters (GLUTs) and MCT-1 Promote MHC-II + Mφ activity in demyelination [138] demonstrated heightened activation of granulocyte colony-stimulating factor (G-CSF)/STAT3 signaling, resulting in atypical EAE characterized by intensified neutrophil activation and elevated generation of ROS [144].The impact of mitochondrial and metabolic dysfunction pathways on neutrophil activities is summarized in Table 5.

Mitochondria in MS DCs
DCs, a type of APCs, are divided into conventional DCs (cDCs) and plasmacytoid DCs (pDCs) [145].pDCs produce a large amount of type I IFN, which plays a role in antiviral innate immunity and exists in the blood as immature cells [146].The number of cDCs and pDCs is significantly upregulated in the CSF of MS patients [147,148].In EAE, pDCs promote Tregs expansion and inhibit the development of CD4 + T cells into Th1 and Th17 cells [149,150].Interferon beta-treated DCs induce the gene expression of IL-12p35 and IL-27p28, which suppress the differentiation of Th17 cells and induce IL-10 secretion through the activation of STAT1 and STAT3 in MS [151].
Research highlights the importance of sirtuin 6 (SIRT6) in regulating several processes such as DNA repair, inflammation, immunology, and energy metabolism, including glucose and lipid metabolism [152].Inhibition of SIRT6 attenuates the onset of EAE by reducing DC migration and activation, potentially mediated via metabolic pathways [153].NADPH oxidase 2 (NOX2) plays a crucial role in regulating the endocytosis of MOG antigen processing in DCs and supports the presentation of MOG antigen to CD4 + T cells through LC3-associated phagocytosis (LAP).Deletion of NOX2 in DCs inhibits the myelin peptide presentation of DCs, preventing the recruitment of CD4 + cells to the CNS in EAE mice [154].Activation of HIF-1α by lactate induces NDUFA4L2 and limits the pro-inflammatory activities and activation of DCs.The inhibition of transcription factor X-box binding protein 1 (XBP1) by NDUFA4L2 in DCs plays a crucial role in controlling pathogenic autoimmune T cells in EAE mice.This regulatory mechanism is achieved through the downregulation of oxidative phosphorylation reprogramming and ROS production [155].
Loss of responsiveness to TGF-β receptor II (TGF-βRII) in monocyte-derived DCs (moDCs) results in increased secretion of IL-12 by moDCs, inducing polarization toward an interferon-gamma-producing Th1 phenotype and exacerbating EAE disease.Mechanistically, upregulated IFN-γ increases Nox-2 and ROS production in moDCs during chronic EAE [156].DMF induces type II DCs by reducing intracellular GSH through the formation of conjugates, leading to upregulation of heme oxygenase-1 (HO-1) levels [157].GSH acts as an effective scavenger of ROS, while HO-1 is a sensitive heat shock protein that may result in higher ROS production [158,159].Additionally, DMF prevents DCs from expressing pro-inflammatory cytokines and costimulatory molecules [160,161].In part, the modulation of the GSH-HO-1 pathway by DMF treatment regulates the oxidative stress during DC maturation and antigen-presenting capacity.These findings suggest that targeting mitochondrial activities of DCs holds promise for the treatment of MS.Table 6 provides a comprehensive overview of the impact

Mechanism of mitochondrial and metabolic dysfunction Neutrophils functions References
Knock-out CXCR2 reduces ROS production through inhibiting Ncf1 and IL-1β production Reduce the inflammatory cytokines [143] Deficiency of Socs3 elevates ROS through activating G-CSF/STAT3 signaling Enhance neutrophil activation [144] Table 6 The impact of mitochondrial and metabolic malfunctioning pathways on the activities of DCs

Mechanism of mitochondrial and metabolic dysfunction DC functions References
Sirt6 inhibition potentially mediates metabolic pathways Reduce migration and activation of DCs [152,153] Knock-out NOX2 in DCs can inhibit T cell-mediated autoimmune neuroinflammation through LC3-associated phagocytosis (LAP) Inhibit myelin peptide presentation of DCs [154] Increased HIF-1α-NDUFA4L2 signaling inhibits X-box binding protein 1 (XBP1) and leads to downregulating OCR and ROS Promote pro-inflammatory activities and activation of DCs [155] Tgfbr2 insufficiency in moDCs upregulates ROS production via NOX2 Secret more IL-12 in moDCs which induces Th1 polarization and chronic inflammatory demyelination [156] DMF partly though GSH-HO-1 pathway regulating oxidative stress Decrease maturation and antigen-presenting capacity of proinflammatory DCs [157,160,161] of mitochondrial and metabolic dysfunction pathways on DC activities.

Conclusion
Metabolic processes such as glycolysis, the TCA cycle, and lipid metabolism have a strong connection with mitochondrial activities.Furthermore, there may exist a reciprocal link among immune metabolism, immune cell activation and mitochondrial function.Metabolic processes and activities play a significant role not only in producing ATP and biosynthetic precursors but also in influencing immunological responses and the functioning of mitochondria in immune cells.However, it is still unclear whether the improvement in abnormal metabolism is primarily caused by the reprogramming of immune activation or mitochondria.
Research has primarily focused on investigating alterations in the morphology, organization, and functionality of mitochondria in immune cells affected by MS.These investigations have explored various mitochondrial metabolic activities, including mitochondrial respiration, ROS production, MMP, TCA metabolic enzymes, and metabolites.Mitochondrial dysfunction manifests with varying severity across different immune cell populations at different stages of MS progression.Furthermore, the presence of abnormal metabolites influences the functionality and maturation of cellular mitochondria, leading to the persistent release of pro-inflammatory cytokines, neurodegeneration, and demyelination, ultimately affecting the progression of MS.Several DMTs targeting mitochondria and metabolites have been shown to enhance the metabolic activity of immune cells in individuals with MS.This intervention has a significant positive impact on the overall disease status, as summarized in Table 7.
However, the complex nature of pathophysiological mechanisms underlying mitochondrial metabolism in MS poses challenges for disease diagnosis and treatment.Unfortunately, current research on precision medicines specifically targeting connections within mitochondrial metabolic pathways is deficient.Further investigation into the mitochondrial and metabolic abnormalities of immune cells, utilizing multi-omics combination techniques and high-dimensional single-cell analysis, may enhance our understanding of the pathophysiology of MS.Table 7 The present study investigates the impact disease-modifying therapy (DMT) medications on several immune cell populations, with a particular focus on the underlying processes including mitochondrial and metabolic dysfunction

DMT drugs Immune cell type Mechanism of mitochondrial and metabolic dysfunction References
Interferon beta T cell Reverse the decreased glycolysis and mitochondrial respiration activity by upregulating the expression of aldolase, HK-1, ENO1, GLUT1, DLAT and DLST [77] DC Suppress the differentiation of Th17 cells and induces IL-10 secretion via activated STAT1 and STAT3 [151] Monocyte Alter monocytic ROS production and mitochondrial ETC-related genes [121,122] Dimethyl

Fig. 2
Fig. 2 Mitochondria in MS CD4 + T cells.CD4 + T cells boost the expression of glucose transporter (GLUT)1, resulting in enhanced glucose uptake and lactate generation, which can be blocked by 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) inhibitors.The deletion of acetyl-CoA carboxylase 1 (ACC1) reduces the synthesis of de novo fatty acids, decreases IFN-γ + Th17 via the glycolytic-lipogenic pathway, and increases Foxp3 + Treg.Pentanoate acts on acetyl-CoA through the mTOR pathway, leading to increased glucose oxidation, secretion of IL-10, and inhibition of IL-17A production.Oleic acid restores the inhibitory state of CD4 + to Treg by promoting fatty acid β-oxidation.Pik3c3 increasing and Nur77 deleting both contribute to the elevation of mitochondrial extracellular acidification rate (ECAR) and oxygen consumption rate (OCR).Upregulation of mEF-G1 caused electron transport chain (ETC) assembly in mitochondria, and then elevated NAD + /NADH ratio.Above three mechanisms enhance Th1 or Th17 differentiation.Additionally, increased mitochondrial ROS can promote Th17 differentiation.Upregulated mitochondrial oxidative phosphorylation (OXPHOS) influences basic leucine zipper transcription factor TF-like (BATF), promoting Th17 differentiation and inhibiting Treg differentiation.DMF as well as the Bim and Bad pathways of phospholipids (PLs) treatment induces increased apoptosis by augmenting mitochondrial ROS production

Fig. 3
Fig.3Mitochondria in MS monocytes.Upregulated pyruvate is converted to enormous lactate by increased expression of lactate dehydrogenase A (LDHA) enzyme in MS monocytes.The 2-deoxy-glucose (2-DG) inhibits glycolysis and reduces glucose uptake as well as lactate production.When monocytes are exposed to cerebrospinal fluid (CSF) from MS patients, there is an increase in the production of intracellular glutamine, pyruvate and extracellular glutamine, lactate and pyruvate.While the production of glutamic acid decreases.The small molecule inhibitor 6877002 inhibits the upregulated ROS, extracellular IL-6, tumor necrosis factor (TNF), and decreases IL-10 through the CD40-TRAF6 pathway.MS patients who respond IFN-β treatment alter in electron transport chain (ETC)-related genes, a mildly decrease ROS, and an improvement in mitochondrial dysfunction